In order to understand cell growth of both normal and abnormal cells, scientists have sought to grow cells in chemically defined culture media. Cells grown in culture media are used for many different purposes and nearly all animal cells in culture require serum for growth. Serum is, however, a complex material containing many substances. Substances present in blood that promote cell growth (cell mitosis) have been termed "growth factors" (mitogens).
Some growth factors affect specific cells while other growth factors cause growth of a broader spectrum of cell types. Numerous scientists have sought means to isolate and purify substances responsible for cell growth. Some substances have been chemically identified and are polypeptide or protein in nature. Purification of such growth factors, usually present in very low concentration in blood, is extremely difficult. Techniques presently available for extraction of growth factors from these complex systems generally are not appropriate for processing large volumes of serum (D. Gospodarowica and J. S. Moran, in Ann. Rev. Biochem., 45, pp. 531-588, 1976). Methodologies available for the fractionation of proteinaceous growth factors from serum were reviewed in my earlier patent, U.S. Pat. No. 4,189,535. This patent teaches adsorption of serum proteins to anionic exchange resins followed by specific elution of growth promotant materials utilizing gradient salt or pH solutions.
One group of growth factors in blood are the somatomedins, a group of circulating peptides, believed to be of liver origin, that appear to be regulated by growth hormone. Somatomedins have been dissociated from larger carrier proteins in acidic environments and it has been shown that active somatomedin molecules of less than 10,000 dalton molecular weight are present in soluble form in ultrafiltrates of human plasma after acidification (pH 2.3) with acetic acid (B. H. Ginsberg et al., J. Clin. Endocrinology and Metabolism 48(1), 43-49, 1979). Although somatomedins can be recovered from plasma protein precipitates formed during Cohn fractionation they are dissociated from the carrier proteins by further acidification. The greater acid solubility of the dissociated somatomedins have been utilized in several methodologies for the collection of these growth promoters from Cohn-precipitated plasma protein. (H. Burgi et al, Biochem, Biophys. Acta. 121, 349-359, 1966; Knut Uthne, Acta Endocrinology (Suppl.) 175, 1-35, 1973; E. Rinderknecht and R. E. Humble, Proc. Natl. Acad. Sci. U.S.A., 73(7), 2365-2369, 1976).
During the blood coagulation process growth factors are released from blood platelets and some researchers have claimed a major portion of serum growth promoters are of platelet origin (R. Ross and A. Vogel, Cell, 14, 203-210, 1978). As a result of the fact that growth factors account for only a small percentage of all serum protein, investigators have isolated platelet growth factors from preparations of lysed washed platelets utilizing ion exchange chromatographic procedures. C. H. Heldin et al., (Exp. Cell Res. 109, 429-437, 1977) described two anionic fractions of 40,000 and less than 10,000 dalton molecular weight, respectively, and a heterogenous cationic fraction of from 25,000 to 35,000 dalton size possessing activity. Ross, et al (in Hormones and Cell Culture, Book A. Eds. G. H. Sato and R. Ross, pp 3-16 (Cold Spring Harbor Laboratory), 1979) isolated a cationic fraction of 10,000 to 30,000 molecular weight. Further processing resolved mitogenic activity to molecules of 16,000 to 18,000 daltons.
Several groups of investigators agree blood contains more than a single growth factor. For this reason, it appears desirable to develop a practical method of separating the diverse group of molecules possessing mitogenic activity from other blood proteins. In addition, there are not only many areas of practical application in which there may be made beneficial substitution of purified growth materials for whole serum or plasma, but the availability of growth material would make possible new areas of application of cell growth regulation. Some of the areas in which the availability of purified growth materials would be highly advantageous include the study of neoplastic diseases, dwarfism, neurological development, cellular immunity, transplantation (tissue grafting), wound healing and replacement of serum for virus vaccine production, for the production of biomolecules (e.g., urokinase, interferon, monoclonal antibodies, etc.), in animal husbandry for increasing and maintaining rapid growth and wherever serum or plasma is required for cell mitosis.
Thus, a purified growth material preparation would be desirable for many applications. The present invention results from a search for an economical procedure suitable for the isolation and purification of the low concentrations of growth promoters that are present in serum or plasma. Such a method will permit processing of moderate to large volumes of starting materials and will produce growth promoters in an active state, free of many of the non-growth promoting substances present in whole serum or plasma.